|Home | About | Journals | Submit | Contact Us | Français|
The inflammatory cytokine TNF-α stimulates several presumed pro-atherogenic signaling events in endothelial cells (ECs), including activation of c-Jun NH2-terminal kinase (JNK) and induction of E-selectin. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a MAP kinase kinase kinase, is required for TNF-mediated JNK activation. TNF activates ASK1 in part by dissociating ASK1 from its inhibitor 14-3-3. Because the risk of atherosclerosis is decreased in regions of steady laminar flow, we hypothesized that laminar flow inhibits proinflammatory cytokine-mediated activation of JNK. Steady laminar flow inhibited both TNF activation of ASK1 and JNK. Inhibition of ASK1 by flow correlated with increased association of ASK1 with 14-3-3. A constitutively active form of ASK1 lacking the 14-3-3-binding site (ASK1-ΔNS967A) was not inhibited by flow. These data establish ASK1 as a target for flow-mediated inhibition of cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator in ECs.
The c-Jun NH2-terminal kinase (JNK) is one of three mitogen-activated protein kinase (MAPK) cascades that are strongly activated by stress signals and proinflammatory cytokines such as TNF and IL-1. The TNF superfamily uses receptors that are devoid of intrinsic catalytic activity (1). Activation of JNK by members of the TNF-receptor family is thought to be mediated by a family of intracellular signaling molecules known as TNFR-associated factors (TRAFs) (2). Six TRAF family members have been described, and different members of this family display distinct receptor-binding specificities. Studies from TRAF2 transgenic and knockout mice demonstrated that TRAF2 is essential for activation of JNK in response to TNF but not IL-1 (3–5). In contrast, TRAF6 specifically mediates IL-1–induced JNK activation by IL-1 receptor (6, 7).
JNK is activated by dual phosphorylation mediated by one of the MAP2Ks (MKK4 and MKK6). MKK, in turn, is activated through phosphorylation by MAP3Ks (including MEKK1, TAK1, and ASK1) (8–10). Some of MAP3Ks such as MEKK1 and TAK1 can activate both NF-κB and JNK cascades and these MAP3Ks are direct targets of TRAF molecules (6, 11, 12). However, some MAP3Ks such as ASK1, appear to be involved in JNK activation only in response to proinflammatory cytokines and stress stimuli (13–15). ASK1 is a 170-kDa protein that functionally is composed of an inhibitory NH2-terminal domain, an internal kinase domain, and a COOH-terminal regulatory domain. The COOH-terminal domain of ASK1 binds to the TRAF domain, and this association is required for ASK1 activation by TRAF2 and TRAF6 (15). A kinase-inactive form of ASK1 (ASK1-K709R) functions as a dominant negative in cytokine-induced JNK activation (13, 15). Deletion of the NH2-terminal 648 amino acids of ASK1 (ASK1-ΔN) leads to constitutive ASK1 kinase activity as it does in other MAP3Ks, indicating that the NH2-terminus contains an inhibitory domain (16, 17).
In addition, several cellular factors, 14-3-3, and thioredoxin, have been reported to inhibit ASK1 activity. Thioredoxin in a reduced form binds to the NH2-terminal part of ASK1 and blocks activation of ASK1 by TNF (16–18); 14-3-3, a phosphoserine-binding molecule, binds to ASK1 specifically via Ser-967 of ASK1 and has been reported to inhibit ASK1-induced apoptosis (19). However, the role of 14-3-3 in TNF-induced ASK1 activation has not been determined.
It is widely accepted that inflammation plays a key role in the pathogenesis and progression of atherosclerosis (20). An important role for JNK in inflammation is supported by many studies. First, JNK is activated by almost all proinflammatory mediators such as TNF, IL-1, LPS, and oxidative stress (10, 21). Second, JNK activation is essential for expression of many proinflammatory molecules such as such as E-selectin, RANTES, IL-12, IL-6, and IL-8 by activating transcription factors including c-Jun and ATF-2 (10, 22–24). This has been demonstrated by either a dominant-negative or antisense approach. For example, overexpression of DN-JNK or antisense JNK cDNA can block IL-6 and IL-8 expression induced by IL-1 (25); overexpression of DN-JNK can block endothelial cell (EC) surface expression in human umbilical vein endothelial cells (HUVECs) (23).
The concept that steady laminar blood flow exerts an atheroprotective effect by modulating EC function is well supported (26, 27). Examples include increased nitric oxide production and decreased expression of cell adhesion molecules such as VCAM-1 and E-selectin (26–28). In this study we demonstrate that activation of JNK, but not NF-κB, by proinflammatory cytokines is attenuated by pre-exposure of EC to laminar flow. To gain insight into the mechanism by which laminar flow inhibits JNK activation by proinflammatory cytokines, we examined the effect of flow on the upstream activators of JNK in TNF signaling pathway. Our results show that flow inhibits ASK1 activity and ASK1-dependent JNK activation by increasing association of ASK1 with its inhibitor 14-3-3.
Mammalian expression plasmids for Flag-epitope labeled wild-type TRAF2 and dominant-negative TRAF2 (dnTRAF2) (29) were provided by D.V. Goeddel (Tularik Inc., South San Francisco, California, USA); wild-type and the kinase-inactive ASK1 by Genhong Chen (University of California, Los Angeles, California, USA); GST-14-3-3 by Anthony J. Muslin (Washington University in St. Louis, St. Louis, Missouri, USA); GST-JNKK1 (MKK4) by Bing Su (M.D. Anderson, Texas, USA), pBIIXLUC plasmid (κB-LUC) (23), which contains two κB sites from the immunoglobulin kappa enhancer, and the JNK-dependent reporter gene system (FR-Luc and FA2-cJun) were from Stratagene (PathDetect Reporting Systems; La Jolla, California, USA). Expression plasmids for ASK1-ΔN (lacking the NH2-terminal domain) and ASK1-ΔNS967A (deletion of the NH2-terminal and mutation at serine 967) were constructed into the Flag-vector.
HUVECs are purchased from Clonetics Corp. (San Diego, California, USA). Human rTNF and rIL-1 are from R&D Systems (Minneapolis, Minnesota, USA) and used at 100 U/ml and 250 U/ml, respectively.
Fluid shear stress in vitro was created by the cone and plate viscometer. Cells were grown on 60-mm dishes coated with gelatin. Upon reaching 95% confluence, fresh media was added, and 2 days later cells were rinsed free of culture media with HBSS (containing, in mM: NaCl 130, KCl 5, CaCl2 1.5, MgCl2 1.0, HEPES 20, pH 7.4),with 10 mM glucose added, and 10% serum, and either maintained in static condition or exposed to flow (fluid shear stress = 12 dynes/cm2) in a parallel plate chamber at 37°C. After varying times of exposure to flow, cells were washed gently with ice-cold PBS (composition, in mM: NaCl 137, KCl 2.7, Na2HPO4 4.3, KH2PO4 1.4, pH 7.3) and cell lysates were for further analysis.
JNK assay was performed as described previously (23) using GST–c-Jun (1–80) fusion protein as a substrate. ASK1 assay was performed using GST-MKK4 as a substrate.
Transfection of HUVECs were performed by DEAE-dextran method, as described previously (23). Luciferase activity followed by renilla activity was measured twice in duplicate using a Berthold luminometer. All data were normalized as relative luciferase light units/renilla unit.
HUVECs after various treatments were washed twice with cold PBS and lysed in 1.5 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.1% Triton X-100, 0.75% Brij 96, 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM sodium pyrophosphate, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM PMSF, 1 mM EDTA) for 20 minutes on ice. For immunoprecipitation to analyze protein interaction in vivo, supernatant of cell lysates were precleared by incubating with normal rabbit serum plus GammaBind plus Sepharose beads on a rotator at 4°C overnight. The lysates were then incubated with the first protein-specific antiserum (e.g., 14-3-3; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) for 2 hours with 50 μl of GammaBind plus Sepharose. Immune complexes were collected after each immunoprecipitation by centrifugation at 13,000 g for 10 minutes, followed by three to five washes with lysis buffer. The immune complexes were subjected to SDS-PAGE followed by immunoblot analysis (Immobilon P; Millipore, Bedford, Massachusetts, USA) with the second protein-specific Ab (e.g., ASK1; Santa Cruz Biotechnology Inc.). The chemiluminescence was detected using an enhanced chemiluminescence (ECL) kit according to the instructions of the manufacturer (Amersham Life Science, Arlington Heights, Illinois, USA). For detection of Flag-tagged proteins (ASK1-ΔN and ASK1-ΔNS967A), anti-Flag M2 Ab (Sigma, St. Louis, Missouri, USA) was used for immunoblot analysis. For detection of HA-tagged proteins (wild-type ASK1), anti-HA Ab (Roche Diagnostics, Indianapolis, Indiana, USA) was used for immunoblot analysis.
The double-stranded oligonucleotide containing a κB consensus site from the immunoglobulin κ gene (Promega Corp., Madison, Wisconsin, USA) was used for electrophoretic mobility shift assays (EMSAs). Preparation of nuclear extracts and EMSA were performed as described previously (23).
To examine the effect of flow on cytokine-induced JNK activation, HUVECs were either maintained in static conditions for 10 minutes or subjected to flow (shear stress = 12 dynes/cm2) for 10 minutes before TNF-α (100 U/ml) stimulation for 15 minutes. TNF-induced JNK activation was determined by an in vitro kinase assay using GST–c-Jun as a substrate, as described previously (23). The results show that pre-exposure to flow significantly attenuated TNF-induced JNK activation. JNK activity was also measured by determining phosphorylation of endogenous c-Jun using Western blot analysis with an Ab against phosphorylated c-Jun (23). A similar effect of flow on TNF-induced JNK activation was observed by this assay. In contrast to our findings, two other laboratories (30, 31) observed JNK activation by flow. “Trivial” explanations for the differences include: we used HUVECs rather than bovine aortic endothelial cells; our cells were several days postconfluent; and our cells were growth-arrested by contact inhibition while the other laboratories used serum deprivation.
To examine whether flow specifically inhibits JNK activation by TNF, we determined the effect of flow on NF-κB activation, a signaling pathway activated in parallel to JNK. We have shown previously that TNF activates mainly the p65/p50 NF-κB complex in HUVECs (23). NF-κB activation was measured by an EMSA using HUVEC nuclear extracts. As shown in Figure Figure1b,1b, pre-exposing HUVECs to flow did not inhibit TNF-stimulated NF-κB activation. These data indicate that flow specifically inhibits JNK activation by proinflammatory cytokines.
To examine the effect of flow on upstream activators of JNK, we measured the activity of ASK1, one of the MAP3Ks involved in activation of JNK, but not NF-κB. ASK1 activity was determined by an in vitro kinase assay using GST-MKK4 (JNKK1) fusion protein as a substrate. As shown in Figure Figure1c,1c, TNF-α (100 U/mL) activated ASK1 maximally at 15 minutes in HUVECs. Pre-exposing HUVECs to flow for 10 minutes significantly inhibited TNF-stimulated ASK1 activity (80% inhibition; n = 3, P < 0.01).
To examine if ASK1 is a specific activator in JNK pathway in HUVECs, we performed JNK- and NF-κB–dependent reporter gene assays by transient transfection in ECs. We used TRAF2 as a control, since we found previously that TRAF2 is involved in TNF-induced activation of both NF-κB and JNK in ECs (23, 32). Overexpression of dominant-negative TRAF2 (DN-TRAF2) blocked TNF-induced expression of JNK-dependent reporter genes (Figure (Figure2a)2a) and NF-κB–dependent gene expression (Figure (Figure2b).2b). In contrast, overexpression of kinase-inactive ASK1 dominant negative (ASK1-K709R) blocked both TNF-induced expression of JNK-dependent reporter genes (Figure (Figure2a),2a), but not NF-κB–dependent gene expression (Figure (Figure2b).2b). These data suggest that ASK1 is specifically required for JNK activation by TNF.
We hypothesized that flow increases the interaction of ASK1 with its inhibitor 14-3-3 to explain how ASK1 activation by TNF is inhibited by flow. Association of ASK1 with 14-3-3 was easily detected in untreated HUVECs (Figure (Figure3,3, control). TNF treatment significantly reduced the interaction of ASK1 with 14-3-3, indicating that TNF activates ASK1, in part, by dissociating ASK1 from 14-3-3. In contrast, flow (using the preconditioning protocol) prevented the TNF-induced dissociation of ASK1 from 14-3-3 (Figure (Figure33).
While thioredoxin binds to the NH2-terminal domain of ASK1 and 14-3-3 binds to the COOH-terminal of ASK1, both inhibit ASK1 activity. An NH2-terminal ASK1 deletion mutant (ASK1-ΔN) no longer binds to thioredoxin and shows increased kinase activity (16, 17). Mutation of serine 967→alanine (ASK1-S967A) renders ASK1 defective in 14-3-3 binding and likely increases ASK1 activity as shown by accelerated ASK1-induced apoptosis (19). We constructed an ASK1 “double” mutant, which lacks the NH2-terminal domain and has a mutation of serine 967→alanine that we termed ASK1-ΔNS967A. To determine whether ASK1-ΔNS967A binds to these two inhibitors, HA-tagged ASK1-WT, Flag-tagged ASK1-ΔN and ASK1-ΔNS967A were transiently transfected into HUVECs, and the interactions of these ASK1 proteins with thioredoxin and 14-3-3 were examined by coimmunoprecipitation assay. As expected, ASK1-WT bound to both thioredoxin and 14-3-3 (Figure (Figure4a).4a). ASK1-ΔN bound to 14-3-3 (lane 1 in Figure Figure4b),4b), but not to thioredoxin (lane 3 in Figure Figure4b).4b). In contrast, ASK1-ΔNS967A failed to bind either thioredoxin or 14-3-3 (lanes 2 and 4 in Figure Figure44b).
To determine the effect of altering ASK1 interaction with 14-3-3, we measured both JNK activity and JNK reporter-gene expression. Expression of ASK1 proteins in transfected ECs was determined by Western blot analysis with an Ab against the COOH-terminal ASK1, which recognized ASK1-WT, ASK1-ASK1-K709R, ASK1-ΔN, and ASK1-ΔNS967A (Figure (Figure5a).5a). The endogenous ASK1 present in ECs was also detected (see lanes 4 and 5). Expression of ASK1-WT, ASK1-ΔN, or ASK1-ΔNS967A increased JNK activity in the absence of TNF as measured by an in vitro kinase assay with GST–c-Jun substrate (Figure (Figure5b)5b) and JNK-dependent reporter-gene expression (Figure (Figure5c).5c). Expression of the kinase-inactive form of ASK1 (ASK1-K709R) had no effect on JNK reporter-gene expression (Figure (Figure5c).5c). These data confirm the function of ASK1 protein in ECs.
Since ASK1-ΔNS967A does not associate with 14-3-3, we hypothesized that ASK1-ΔNS967A activity would not be inhibited by flow. To test this hypothesis, ECs were transfected with Flag-tagged ASK1-ΔN or ASK1-ΔNS967A constructs. Because ASK1-ΔN and ASK1-ΔNS967A are constitutively active, ECs were exposed to flow in the presence of TNF 24 hours after transfection. Flag-tagged ASK1-ΔN or ASK1-ΔNS967A were immunoprecipitated by anti-Flag, and their activities were determined by an in vitro kinase assay using GST-MKK4 as a substrate. As shown in Figure Figure6a,6a, ASK1-ΔN expression was not altered by flow, but its activity, as measured by phosphorylation of GST-MKK4, was significantly inhibited by flow (Figure (Figure6,6, b and c). In contrast, the activity of ASK1-ΔNS967A was not inhibited by flow. These data suggest that flow inhibits ASK1 activity by regulating association of ASK1 with its inhibitor 14-3-3.
Ser-967 of ASK1 is part of a motif RxSxxP reported to bind 14-3-3; therefore, we hypothesized that TNF and flow alter ASK1 phosphorylation at Ser-967 to regulate the interaction of ASK1 with 14-3-3 (Figure (Figure7a).7a). To test this hypothesis, HA-ASK1 transfected into ECs. ECs were exposed to flow or TNF 24 hours later. To measure pSer-967 of ASK1 we developed an in vitro 14-3-3 binding assay. Phosphorylated ASK1 Ser-967 was first bound to GST-14-3-3, followed by Western blot analysis using anti-HA. This is different from the coimmunoprecipitation assay described in Figure Figure3,3, which measured preexisting complexes of ASK1 and 14-3-3. We first examined function of GST–14-3-3 proteins. The results show that ASK1 specifically binds to wild-type 14-3-3, but not to a mutant 14-3-3 (K49A) defective in phosphoserine-binding (33) (Figure (Figure7b).7b). Consistent with our observation in Figure Figure4,4, ASK1-ΔN, but not ASK1-ΔNS967A, bound to GST–14-3-3 (data not shown). These data suggest GST–14-3-3 specifically binds to phosphorylated ASK1 Ser-967 in an vitro binding assay. Therefore, we use this GST–14-3-3 binding assay to determine effects of TNF and flow on phosphorylation of ASK1 Ser-967. Our results show that TNF decreased the amount of phosphorylated ASK1 leading to reduced binding on GST–14-3-3 (Figure (Figure7b).7b). In contrast, flow prevents dephosphorylation of ASK1 at Ser-967, resulting in increased 14-3-3 binding (Figure (Figure7c).7c). These data suggest that TNF and flow regulate ASK1 activity by modifying the phosphorylation status of ASK1 Ser-967.
The major finding of the present study is that one of the mechanisms by which steady laminar flow inhibits proinflammatory events is via inhibition of ASK1. Based on findings in the present study, we propose a model in which TNF activates ASK1 (in part) by dissociating ASK1 from its inhibitor 14-3-3, while steady laminar flow inhibits TNF-induced ASK1 and JNK activation by preventing the release of ASK1 from 14-3-3 (Figure (Figure7).7). Because steady flow may exert atheroprotective effects by inhibiting inflammation, these results imply an important role for 14-3-3 in atherosclerosis.
While some MAP3Ks such as TAK1 and MEKK1 are involved in both JNK and NF-κB activation, our data indicate ASK1 is one of the major MAP3Ks specifically associated with JNK activation in ECs. It remains to be determined whether other MAP3Ks are regulated by TNF and flow in ECs.
The activation mechanisms for MAP3Ks in “the stress-activated” MAPK pathways remain largely unknown. A role for reactive oxygen species (ROS) has been proposed (17). Specifically, hydrogen peroxide activates ASK1, and TNF-induced activation of ASK1 is inhibited by antioxidants (18). Thioredoxin, a redox-sensing protein, associates with ASK1 in its reduced form. Recently, it has been shown that TNF (as well as overexpression of TRAF2) stimulates the production of ROS (16). Activation of ASK1 by TNF requires the ROS-mediated dissociation of thioredoxin followed by binding of TRAF2 and subsequent ASK1 dimerization (16). The fact that flow also increases ROS in ECs (34, 35) suggests that thioredoxin is not a critical mediator of flow in regulating ASK1 activity. This is supported by our data that activity of the thioredoxin binding–deficient mutant (ASK1-ΔN) is still inhibited by flow.
We found that 14-3-3 binds to ASK1 in unstimulated HUVECs, indicating that ASK1 is constitutively phosphorylated at Ser-967 and forms a preexisting complex with 14-3-3 as described previously (19). A 14-3-3 molecule with two mutations (R56A and R60A) is deficient in its ability to bind phosphoserine-containing peptides and functions as a dominant-negative 14-3-3 signaling molecule (33). Recently Xing et al. showed that expression of DN-14-3-3 (R56A/R60A) activated ASK1 and JNK, likely by releasing ASK1 from inhibition by endogenous 14-3-3 (36). This is consistent with our finding that proinflammatory cytokines stimulate release of ASK1 from 14-3-3, whereas flow inhibits the release. Currently the mechanism by which cytokines and flow regulate association of ASK1 with 14-3-3 is not understood. However, our data suggest that phosphorylation of ASK1 Ser-967 (which is the 14-3-3 binding site) is the critical step. We suggest that TNF activates a phosphatase that diminishes 14-3-3-ASK1 interaction, whereas flow inhibits this phosphatase to enhance formation of 14-3-3-ASK1 complex (Figure (Figure7).7). Our finding that activity of the 14-3-3 binding-deficient mutant (ASK1-ΔNS967A) is not inhibited by flow further supports this model. Experiments to identify this phosphatase are underway.
In summary, these data establish the ASK1-JNK signaling cascade as a target for flow-mediated inhibition of proinflammatory cytokine signaling and indicate a novel role for 14-3-3 as an anti-inflammatory mediator. While the roles of ASK1 and JNK in atherogenesis have only been studied to a limited extent, this study suggests that inhibition of ASK1-JNK pathway may provide a valid approach for antiatherosclerotic therapy.
We thank D.V. Goeddel for TRAF2 and DN-TARF2 constructs; Genhong Chen for wild-type and the kinase-inactive ASK1; Anthony J. Muslin for GST-14-3-3; Stephanie Lehoux for GST-14-3-3 K49A; Bing Su for GST-JNKK1 (MKK4). We also thank B.C. Berk lab members for assistance and discussions. This work was supported by NIH grants 1R01HL-65978-01 to W. Min and 5P01HL-18645 to B.C. Berk.
Yingmei Liu and Guoyong Yin contributed equally to this work.